CURRENT COMMENT Transplantation Immunity Hy FHEDEHK C. McDur~its F HUM THE BlBLlCAL command “if- thine eye offend thee, pluck it out and cast it from thee” has grown the branch of medicine known as surgery. Its development has been based on the principle that many disease processes are localized in single organs, the removal of which wiil result in the restoration of an invalid to health. That many diseases are not amenable to this form of treatment is usually due to their being so widespread (e.g. disseminated lupus) that one hardly knows where to begin or to their being localized to an organ (e.g. the heart) which cannot be so easily dispensed with as the appendix or gallbladder. The dream of being able not only to remove tissues that are beyond repair but to replace them with something almost as good as new has been with the surgeon for many centuries, but only in the past few years has any possibility of its becoming a reality developed. The long entertained idea that the rejection of homografts was an immunological phenomenon was largely established by the work of P. B. Medawar and his group at the University of London during the 1940’s and early 1950s. The final and most direct proof was the demonstration by Mitchison in 1954l that transplantation immunity could be transferred from one mouse to another of the same inbred strain by means of lymphoid cells. The use of inbred animals by workers in the field of transplantation immunity had been pioneered by Little and later by Snell in Bar Harbor and by Gorer in London, beginning in the 1930’s. The establishment of the genetic basis for transplantation immunity by these latter workers at once clarified the problem and at the same time demonstrated the magnitude of its eventual solution. The additional demonstration by Medawar and his associates, notably Billingham and Brent, of the phenomenon of immunological tolerance completed the scientific groundwork on which present-day work in this field is based. For example, after transferring spleen cells of CBA mice into A line mice within 24 hours of birth they were able to show that the CBA cells persisted into adult life and that these A mice would accept grafts from CBA mice without rejecting them-an example of artificial chimerism.2 This tolerance did not appear to be organ specific in any way Chronic renal failure was a logical disease in which to attempt to use this new knowledge because of the usual hopelessness of the situation, the relatively straightforward technical problem, the availability of the‘ artificial kidney and the fact that the recipients’ own kidneys could be left in place. The reports of Hume et al. in 1952 and 1955 in this country3 as well as those of Kiiss et al. in 19514 and of Michon et al. in 19535in France marked the beginnings of serious efforts to accomplish successful renal homotransplantation in man. Numerous technical problems have now been overcome, and progressively longer survivals have been achieved, resulting, in a few rare 87 AniHRITrs AND RHEUMATISM, V O L . 7 , NO. 1 ( FEBAUAIiY ), 1984 88 FREDERICK C . MCDUFFIE instances, of almost complete reversal of uremia and hypertensive vascular disease. The major problems have been lack of knowledge about the chemistry and genetics of the antigens responsible for homograft rejection, the lack of quantitative immunologic methods for following the phenomenon and our inability to completely understand the mechanism of tolerance. Most of our knowledge about histocompatibility genes comes from work done with inbred mice, a subject which has recently been very lucidly reviewed by Sne1l.O At least 14 loci are involved, and it is possible that the true number is much greater. Four of these loci have been accurately mapped, and a fifth is known to be present on the Y or male sex chromosome. From studies performed on coisogenic strains (inbred strains which differ only at a single histocompatibility locus) it has been possible to show that the antigens governed by different loci differ in their ability to produce transplantation immunity. The antigens controlled by the well known H-2 locus appear to be the most highly antigenic since differences at this locus are reflected by rapid rejection of grafts. The antigen controlled by the locus on the Y chromosome is very weak indeed, as prolonged survival of grafts from males to females of the same strain often occurs. The antigens governed by the H-1, H-3 and H-4 loci are intermediate in their behavior. Much less is known about the chemistry of these antigens. Because of the number of genetic loci known to be involved it seems likely that several proteins and polysaccharides and possibly nucleoproteins may serve to stimulate transplantation immunity. Billingham, Brent and Medawar7 prepared a soluble extract from spleen cells which could produce accelerated graft rejection if given in a single sensitizing dose prior to grafting. This material could be inactivated by periodate and also by a crude enzyme extract of Trichomonas foetus known to inactive blood group substances. Kandutsch and Stimpfling* and Vranken-Paris et al.9 have each isolated an electrophoretically and ultracentrifugally homogeneous lipoprotein containing H-2 antigenicity as measured by ability to cause accelerated rejection of skin grafts and to absorb or inhibit hemagglutinins appearing in the serum after grafting. Compounding the problem created by the multiplicity of transplantation antigens is uncertainty about the basic nature of transplantation immunity. Still unsettled is the question as to whether it is mediated by classical antibody or whether it represents a form of delayed sensitivity, presumably produced directly by sensitized cells. It was recognized in 1937 by GorerlO that the serum of mice which had rejected an A strain sarcoma would agglutinate strain A erythrocytes. The H-2 locus has been shown to govern the production of an antigen present in red cells, antibody to which is responsible for this agglutination as well as for the phenomenon of immunologic enhancement. By this term is meant the prolongation of graft survival resulting from pretreatment with antigen (first produced with lyophilized tumor tissue by Casey in 194111).Antibodies have been shown to exert a direct toxic effect against cells in tissue culture, particularly in the presence of complement. Circulating antibodies, however, most often cannot be detected when grafts involving TRANSPLANTATION IMMUNITY 89 other antigens than H-2 are rejected and, when they are present, their titer does not always correlate directly with the rate of rejection. It has not been regularly possible to transfer homograft immunity with serum except against certain transplantable tumors. Billingham et al. have shown in mice’ and Lawrence et al. in humans12 that the rejection of homografts is accompanied by the development of delayed skin sensitivity to extracts of donor tissue. There is unfortunately no good method for measuring delayed sensitivity quantitatively. The demonstration that transplantation immunity could be transferred with viable lymphoid cells does not favor either hypothesis since circulating antibody against graft antigen may be produced by such cells but removed rapidly from the circulation by the graft. Work by Algire in 195413 with transplants contained in chambers permeable to antibody but not to whole cells tended to show that such grafts were protected from the rejection phenomenon. However, transplantation immunity can be transferred by lymphoid cells placed in such chambers14 and Algire himself’: later showed that after more intensive immunization some grafts are not protected in these chambers. Stetsonlo believes that antibody is responsible for the rapid rejection of grafts that never become vascularized (“white grafts”) whereas classical rejection is a manifestation of delayed sensitivity. At any rate the lack of a measurable immune system known to correlate with the rejection of a graft has proved a serious obstacle to the transplanter. To obviate some of the difficulties in humans, Murray et aL17 use the triple skin graft technique to estimate the degree of relationship between the donor and recipient prior to renal transplantation. Skin from the prospective recipient is grafted onto a test subject. Fourteen days later skin from prospective donors is grafted onto the same subject. Presumably the graft sharing the most antigens with the intended recipient will be rejected the most rapidly. Brent and Medawar18 have performed delayed skin tests employing viable blood leukocytes for estimating the relative amount of sharing of transplantation antigens. In view of these obstacles to successful homotransplantation in humans, attention in the past few years has turned to attempts to modify the immune response so that the strong transplantation barriers may be crossed more easily. The effects of x-irradiation on the suppression of antibody production had been previously studied in great detail and it was soon learned that artificial tolerance to homografts could be produced by this agent. Antimetabolites such as 6-mercaptopurine have been shown to exert a similar effect and have become more widely used than x-ray since they are easier to administer, and the effect on rejection can be more carefully titrated by careful adjustment of dosage. Their ability to prolong survival of renal homografts in dogs was reported by Zukoski et al. in 19601@and they have been extensively used in both animal and human transplantation work since that time. In the doses used they do not seem to have much impaired the ability of the host to defend himself against infection although this has not been carefully measured. These agents have very little effect on the secondary antibody response so that they may not affect the immune response to common bacterial pathogens. It is not 90 FREDERICK C . MC DUFFIE clear, however, whether their inhibitory effect on the rejection phenomenon is truly due to immunologic suppression or whether the character of the inflammatory response is in some way modified. 6-mercaptopurine can produce a marked inhibition of the inflammatory response, even in the absence of leukopenia, as Page, Condie and Good have recently demonstrated.20 In a quantitative study of immunological tolerance reported last year, Brent and Gowland21measured the relationship between age of the recipient and dose of transplanted cells in the production of tolerance in newborn mice. The number of cells necessary increased for the first twelve days of life, but by thirteen days of age tolerance could no longer be produced with even a very large single dose of cells. It could however, deveIop after frequent repeated doses of donor cells given over a period of time. It seems that both neonatal and adult immunologic unresponsiveness are essentially the same phenomenon and depend on the proper relationship between size of the dose of antigen and size (or capacity) of the recipient’s immunologic potential. From these observations it would appear that an increase in the size of the graft beyond a certain point might depress the rejection response in a human recipient whose immunologic defenses had been partially suppressed by x-ray, antimetabolites or alkylating agents. Within certain limits an increase in graft size increases the magnitude of the immune response, but above a certain critical size tolerance might be produced in a host whose immunologic capacity had been reduced. That mere size alone cannot induce tolerance is evident from the graft versus host reaction which often occurs after the introduction of a relatively small number of foreign spleen or lymphoid cells into a tolerant host-for example the introduction of parental spleen cells into an F-1 hybrid. Another approach worthy of further trial in humans is the pretreatment of recipients with lyophilized donor cells or with a soluble extract of such cells, prior to grafting. Billingham and Silvers22were able to cross the relatively weak Y antigen barrier in mice using such a tissue extract. A similar suppressive effect on the development of experimental allergic encephalomyelitis has been seen following pretreatment with a soluble brain antigen.23 This latter suppression may also be produced by the passive administration of serum containing complement fixing antibodies against brain tissue.24 The relative roles of immunologic enhancement and immunologic tolerance in the protection afforded by pretreatment with antigen warrant further study. In summary it is dear that further advances in the ability to transplant organs successfully are not limited by the technical ability of surgeons. Temporary survival of transplanted lung25and liver have recently been reported in humans. The rapid advances in our understanding of the mechanism of homograft rejection in the past 15 years and the beginning of promising efforts with human grafts hold o u t hope for the future. As is so frequently true, the chief obstacles are quantitative as well as qualitative-the large number of antigens involved and the lack of quantitative methods for measuring the immunologic response. 91 TRANSPLANTATION IMMUNITY REFERENCES 1. Mitchison, N. A.: Passive transfer of transplantation immunity. Proc. Roy. SOC.B 142:72, 1954. 2. Billingham, R. E., Brent, L., and Medawar, P. B.: Quantitative studies on tissue transplantation immunity. 111. Actively acquired tolerance. Phil. Trans. B 239:357, 1956. 3. Hume, D. M., Merrill, J. P., and Miller, B. F.: Homologous transplantation of the human kidneys. J. Clin. Invest. 31:640, 1952. -, - -, and Thorn, G. W.: Experiences with renal homotransplantation in the human. Report of nine cases. J. Clin. Invest. 34:327, 1955. 4. Kiiss, R., Teinturier, J., and Milliez, P.: Qnelqiies essai\ de greffe de rein chez l’honime. MBm. Acad. de Chir. 77: 755, 1951. (Cited in 3 above.) 5. Michon, L., Hamburger, J., Oeconomos, N., Delinotte, P., Richet, G., Vaysse, J., and Antoine, B.: Une tentative de transplantation r6nale cliez I’homme: aspects m6dicaux et biologiques. Presse med. 61:1419, 1953. 6. Snell, G. D.: The immunology of tissue transplantation in Conceptual Advances in Immunology and Oncology, Hoeber Medical Division, New York 1963, p. 323. 7. Billingham, R. E., Brent, L., and Medawar, P. B.: The antigenic stimulus in transplantation immunity. Nature 178:514, 1956. -, -, and -: Extraction of antigens causing transplanation immunity. Transpl. Bull. 5:377, 1958. 8. Kandutsch, A. A., and Stimpfling, J. H.: Partial purification of tissue isoantigens from a mouse sarcoma. Transplantation 1:201, 1963. 9. Vranken-Paris, M., Lejeune, G., Castermans, A., Dieu, H. A,, and Haenen-Severyns, A. M.: Essais de s b a ration et d’identification des antighes de transplantation. Bull. Soc. Chim. Biol. 44:365, 1962. 10. Gorer, P. A.: The genetic and antigenic basis of tumor transplantation. J. Path. and Bact. 44:691, 1937. 11. Casey, A. E.: Experiments with a mate~ rial from the Brown-Pearce tumor. Cancer Research 1:134, 1941. 12. Lawrence, H. S., Rapapart, F. T., Converse, J. M., and Tillett, W. s.: The transfer of homograft sensitivity (accelerated rejection) with DNasetreated leukocyte extracts in man. Ann. N. Y. Acad. Sci. 87:223, 1960. 13. Algire, G. H.: Vascular reactions of normal and malignant tissues in vivo. VII. Observations on vascular reactions in destruction of tumor homografts. J. Natl. Cancer Inst. 15:483, 1954. -, Weaver, J. M., and Prehn, R. T.: Growth of cells in vivo in diffusion chambers. I. Snrvival of homografts in immunized mice. J. Natl. Cancer Inst. 15493, 1954. 14. Najarian, J. S., and Feldman, J. D.: Passive transfer of transplantation immnnity. I. Tritiated lymphoid cells. 11. Lymphoid cells in millipore chambers. J. Exp. Med. 115:1083, 1962. -, and -: Passive transfer of contact sensitivity by tritiated thymidine labeled lymphoid cells. ibid. 117:775, 1963. 15. Algire, G. H.: Growth inhibition of homografts of a plasma-cell neoplasm in cell-impenetrable diffusion chambers placed in hyperimmunized mice. J. Natl. Cancer Inst. 23:435, 1959. 16. Stetson, C. A.: The role of antibody in the rejection of liomografts. in Mechanisms of hypersensitivity, Edited by J. H. Shaffer, G. A. LcGrippo and M. W. Chase, Little Brown and Company, Boston 1959, p. 569. 17. Murray, J. E., Merrill, J. P., Harrison, J. H., Wilson, R. E., and Dammin, G. J.: Prolonged survival of human-kidney homografts by immunosuppressive drug therapy. New Eng. J. Med. 268:1315, 1963. 18. Brent, L., and Medawar, P. B.: Tissue transplantation: A new approach to the “typing” problem, Brit. Med. J. ii:269, 1963. 19. Zukoski, C . F., Lee, H. M., and Hume, D. M.: The prolongation of functional survival of canine renal homografts by 92 FREDERICK C . MCDUFFIE 6-rnercaptopurine. Surg. For. 11:470, 1960. 20. Page, A. R., Condie, R. M., and Good, R. A.: Effect of 6-mercaptopurine on inflammation. Am. J. of Path. 40619, 1962. 21. Brent, L., and Gowland, G.: On the mechanism of immunological tolerance. in Conceptual Advances in Immunology and Oncology, Hoeber Medical Division, New York, 1963, p. 355. 22. Billingham, R. E., and Silvers, W. K.: Studies on tolerance of the Y chromosome antigen in mice. J. Immunol. 85:14, 1960. 23. Shaw, C.-M., Fahlberg, W. J., Kies, M. W., and Alvord, E. C.: Suppression of experimental “allergic” encephalomyelitis in guinea pigs by encephalitogenic proteins extracted from homologous brain. J. Exp. Med. 111:171, 1960. 24. Paterson, P. Y., and Martin, H. S.: Suppression of allergic encephalomyelitis in rdts by means of antibrain seruni. J. Exp. Med. 117:755, 1963. 25. Hardy, J. D., Webb, W. R., Dalton, X I . R. Jr., and Walker, G. R. Jr : Lung homotransplantation in inm. J.A.hl.A.186: 106.5, 1963. Fredevic C . McDufie, M.D., Associate Professor of Medicine and Assistant Professor of Microbiology, Unioersity of Misslssippi Medical Cen.ter, Jackson, Mississippi.